Our overall hypothesis is that critical frequencies of unique subsets of T. cruzi-specific CD4+ and CD8+ T cells are required to provide optimal mucosal and systemic protection against this protozoan parasite. We have demonstrated that T. cruzi-specific Type 1 responses provide optimal protection against both mucosal and systemic T. cruzi challenges, and that the unique T. cruzi trans-sialidase (TS) antigen can induce both mucosal and systemic protection. However, a large superfamily of TS homologues are encoded by the T. cruzi genome, are expressed during mammalian infection and may have evolved as an immune evasion strategy facilitating parasite persistence. In addition, although our TS vaccines protect against death and reduce mucosal replication during acute T. cruzi infection, chronic infection is not prevented. Therefore, it is critically important to determine the detailed effects of T. cruzi infection on clonal populations of TS-specific T cells. We have identified H-2d-restricted CD4 and CD8 epitopes within the protective TS antigen, shown that a single pair of CD4 and CD8 TS epitopes can induce protective immunity in BALB/c mice, and generated TCR transgenic and retrogenic T cells reactive with these protective CD4 and CD8 TS epitopes. Thus, we are uniquely poised to characterize at the molecular level the frequencies, phenotypes, trafficking molecules and immunological mechanisms involved in the interactive development of memory CD4+ and CD8+ T cells required for long-term protection against an important human pathogen, and to identify the detailed effects of T. cruzi infection on these T cells. We will focus on 3 major aims: aim 1) to molecularly define the CD4+ T cell number, phenotype and mechanisms required to induce and maintain T. cruzi mucosal and systemic protection without induction of immunopathology;aim 2) to molecularly define the CD8+ T cell number, phenotype, and mechanisms required for optimal T. cruzi mucosal and systemic protection without induction of immunopathology;and aim 3) to determine whether mucosal and systemic T. cruzi vaccines induce long term central memory T cells that retain differential activation and/or lymphocyte trafficking profiles. Aims 1-3 will utilize TS vaccination models and focus on responses induced by a well- defined pair of CD4 and CD8 epitopes sufficient for the induction of protective immunity. Tetramer reagents, TCR transgenic/retrogenic mice, and well characterized TS vaccination protocols will facilitate this work. Aim 3 will use transcriptional profiling to identify molecules important for mucosal protection, and determine whether long term central memory T cells can retain differential trafficking profiles. Molecular targets will be experimentally validated in chemotaxis, adhesion, adoptive transfer and/or signaling assays. Overall, the proposed work will provide important new practical and basic immunological information relevant for developing vaccines against many human pathogens.